PROJECT SUMMARY Nerve damage following surgery is a continued morbidity experienced by ?600,000 patients annually in the US alone. Currently no clinically approved method exists to enhance direct nerve visualization in the surgical suite. Nerve-sparing prostatectomy is a compelling clinical example of significant patient morbidity, where nerve damage is reported in ?60% of patients resulting in incontinence and impotence. Development of a near infrared (NIR) fluorophore that specifically highlights nerve tissue in the operating room would have direct clinical translation to nerve sparing prostatectomy through the FDA approved fluorescence channel in the da Vinci surgical robotic system (Intuitive Surgical), which is currently utilized for ?80% of robotic assisted nerve sparing radical prostatectomies (RARP) and will be used for swine imaging herein. Previous work from our lab synthesized libraries of nerve-specific fluorophores that bind to all nerve tissue when administered systemically. However, the prostate structure is highly innervated and systemic labeling of all nerve structures is less desirable compared to directly labeling the neurovascular bundle that contains the nerves responsible for function. A direct fluorophore labeling methodology will be validated herein for in vivo nerve imaging in rodent and swine models, where generation of nerve signal to background ratio (SBR) equivalent or better than systemic administration will be achieved. Nerve imaging in the prostate presents an added challenge as the organ is surrounded by adipose tissue. We have shown that fluorophores with physiochemical properties in the range to cross the blood nerve barrier (BNB) also often exhibit lipophilicity and adipose accumulation upon systemic administration. We have established that co-administration of a spectrally distinct, non-specific fluorophore can be used to differentiate specific and non-specific tissue uptake. This strategy will be investigated, where we will examine the co-administration of spectrally distinct NIR adipose- and nerve-specific fluorophores for their ability to enhance nerve SBR as compared to administration of a nerve-specific fluorophore alone. In the proposed work, we will synthesize a focused oxazine library modeled after our lead, nerve-specific, red-shifted Oxazine 4 fluorophore. The 400 synthesized oxazine fluorophores will be characterized for spectral properties and tissue biodistribution using our previously developed assays. Two direct administration strategies will be investigated in rodent and swine nerves using Oxazine 4 as a model nerve-specific fluorophore to determine the strategy that provides the highest nerve SBR. Dual administration of spectrally distinct oxazine nerve- and adipose-specific fluorophores will be tested to enhance nerve SBR over nerve-specific fluorophore administration alone. NIR oxazine fluorophores will be formulated for in vivo studies, where the optimized direct administration strategy will be used in rodents. Quantified rodent nerve SBR will be used to select the formulated NIR oxazines to scale to swine studies, which will be conducted in collaboration with Intuitive Surgical using the fluorescence channel in a da Vinci surgical robot.